Purified capsular polysaccharides of bacteria have been used to prepare vaccines against the cognate bacteria, but the resulting immune responses have often been less satisfactory than desirable, especially in very young children or individuals with immature or deficient immunological systems. The Streptococcus pneumoniae type 14 capsular polysaccharide, for example, fails to provoke an immune response in infants, thus making this polysaccharide ineffective by itself in providing protection against the serious pediatric medical problems caused by Streptococcus pneumoniae type 14 bacteria, see, for example, Douglas et al., J. Infect. Diseases, 148, 131-137 (1983) and Laurence et al., Am. J. Diseases of Children, 137, 846-850 (1983). Enhancement of the immunogenicity of these polysaccharides may often be accomplished by combining them with proteins, Schneerson et al., Infection and Immunity, 45, No. 3, 582-591 (1984) (discussing conjugation of Streptococcus pneumoniae type 6A).
Care must be exercised in the selection of the protein which is to be combined with these polysaccharides, however. Certain proteins (e.g., pertussinogen) are non-specific stimulators of the immune system in infants. These proteins can, to a degree, enhance the immune response to polysaccharide antigens, but unfortunately, such non-specific activation leads to unwanted biological effects (i.e., reactogenicity). The much preferred specific enhanced immune responses to these polysaccharide antigens can be achieved in infants by "conjugating" these polysaccharides to appropriate proteins, as first reported by W. F. Goebel and O. T. Avery in 1929 (J. Exptl. Medicine 50, 521-531 (1929)).
The means of combining the polysaccharide and protein must also be carefully considered. If, as is believed, the immunological enhancement is realized as a result of the molecular proximity of the polysaccharide determinants to the protein "carrier" determinants, these moieties should not easily separate in the biological system. Non-covalent complexes, arising from the polyanioic character of many polysaccharides and the polycationic character of "carrier" proteins, may stimulate immune responses, but these complexes are chemically labile and the resultant immune responses appear to show T-cell independency. By contrast, covalent conjugates of polysaccharides and protein would possess much greater chemical stability and could demonstrate T-cell-dependent immune responses.
Covalent polysaccharide-protein conjugates have been claimed in the literature, but the exact nature of the covalent linkage has not been proven or quantified since the only assay for covalency has been activity in vivo. In addition, the processes disclosed in the literature have poor reproducibility. Haemophilus influenzae type b and Streptococcus pneumoniae type 6A polysaccharides were reacted with cyanogen bromide, then with adipic acid dihydrazide, then "coupled" with tetanus toxoid or horseshoe crab hemocyanin proteins in Schneerson et al. J. Exptl. Med., 152, 361 (1980) and Infection and Immunity, 40, 245 (1983). Pneumococcal type 19F polysaccharide was coupled to bovine serum albumin directly by forming imines (Schiff bases) from the reducing ends of the polysaccharides and the pendant amine groups (i.e., lysines) of the protein, then reducing these imines with sodium cyanoborohydride (Lin et al., Immunology, 46, 333 (1982)).
Additionally, polysaccharides linked to diazotized aromatic amines were coupled to the protein's tyrosines in K. K. Nixdorff et al., Immunology 29, 87 (1975) and polysaccharides linked to aromatic amines were converted to isothiocyanates, which were then linked to the pendant amino groups of the protein's lysine in S. B. Svenson and A. A. Lindberg, J. Immunolog. Methods 25, 323 (1979). In each case, however, the resulting conjugate was characterized only by its gel permeation chromatographic behavior. In still another example (S. Nutani et al., Infection and Immunity 36, 971 (1982)), the polysaccharide, pullulan, was activated with cyanuric chloride, then reacted with tetanus toxoid. In this case, the conjugates were characterized by electrophoresis and only shown to be different from the starting materials.
In none of these cases was covalency demonstrated other than by the implications of an aggregated molecular weight, thereby confusing covalency with the natural interaction of macromolecular species, with and without charges, in molecular complexes, as these complexes will also give an aggregate molecular weight.
In copending application, U.S. Ser. No. 608,738, filed May 10, 1984, (and incorporated herein by reference) covalently-modified polyanionic polysaccharides and proteins have now been shown, together with covalent conjugates of such polysaccharides linked by abigeneric spacer with immunogenic bacterial membrane or other proteins and methods of preparing these and of confirming the covalency of the linkage between polysaccharides and proteins. Using the methodology of this reference, it is now possible to produce chemically-stable polysaccharide-protein conjugates which demonstrate T-cell dependency and which would be useful as vaccine components for eliciting protective serum antibodies to, particularly, the cognate bacteria of the polysaccharides used. This methodology is, however, only useful for covalently-modifying polyanionic polysaccharides and has not been useful with intractable polysaccharides which are insoluble or only semi-soluble in organic solvents or salt solutions.
It was, therefore, an object of this invention to develop a method for solubilizing neutral polysaccharides and covalently-modifying these neutral polysaccharides in preparation for preparing chemically-stable polysaccharide-protein conjugates. It was also an object of this invention to link neutral polysaccharide determinants to protein "carrier" determinants, in chemically-stable conjugates, such that the molecular proximity of these moieties could be maintained in biological systems in order that these conjugates would be useful as components in a mono- or polyvalent vaccine for eliciting protective serum antibodies to certain bacteria, particularly the cognate bacteria of the polysaccharides used. It was a further object of this invention to develop methods of treatment employing these conjugates in immunologically-effective vaccines for use against, e.g., meningitis and otitis media.